Question

REFLECT AND APPLY The enzyme lactate dehydrogenase catalyzes the reaction \\[ \text { Pyruvate }+\mathrm{NADH}+\mathrm{H}_{\mathrm{y}}^{+} \rightarrow \text { lactate }+\mathrm{NAD}_{\text {Shawn }}^{+} \text {O. Far } \\] NADH absorbs light at \(340 \mathrm{nm}\) in the near-ultraviolet region of the electromagnetic spectrum, but \(\mathrm{NAD}^{+}\) does not. Suggest an experimental method for following the rate of this reaction, assuming that you have available a spectrophotometer capable of measuring light at this wavelength.

Short answer

Monitor the absorbance at 340 nm using a spectrophotometer to follow the rate of the reaction by observing the decrease in NADH concentration.

Step-by-step solution

Understand the Reaction

The reaction involves the conversion of pyruvate and NADH to lactate and NAD^+ catalyzed by the enzyme lactate dehydrogenase. NADH absorbs light at 340 nm while NAD^+ does not.

Identify the Measuring Tool

Use a spectrophotometer capable of measuring absorbance at 340 nm, which is sensitive to NADH but not NAD^+.

Measure Initial Absorbance

Prepare a reaction mixture containing pyruvate, NADH, and the enzyme lactate dehydrogenase. Measure the initial absorbance at 340 nm, which corresponds to the concentration of NADH.

Monitor Decrease in Absorbance

Monitor the absorbance at 340 nm over time. A decrease in absorbance indicates the conversion of NADH to NAD^+, thus providing a measure of the reaction rate.

Calculate Reaction Rate

Calculate the reaction rate by determining the rate of change of absorbance over time, using the Beer-Lambert law to relate absorbance to concentration.

Key concepts

lactate dehydrogenase

Lactate dehydrogenase (LDH) is an enzyme that plays a crucial role in the conversion of pyruvate to lactate. This reaction is essential in cellular respiration, especially during anaerobic conditions when oxygen levels are low. LDH can be found in various tissues throughout the body, including the liver, heart, and muscles.
The reaction it catalyzes involves the interconversion of pyruvate and lactate with the concurrent oxidation and reduction of NADH to NAD+. Enzymes like LDH speed up chemical reactions by lowering the activation energy, making it easier for reactions to occur.
Understanding LDH activity can be particularly useful in clinical settings. Elevated LDH levels in the blood, for instance, can indicate tissue damage or disease. In a laboratory setting, LDH activity is often measured to study metabolic processes, such as glycolysis and fermentation.

NADH absorption

NADH, or nicotinamide adenine dinucleotide (reduced form), plays a key role in energy production within cells. NADH absorbs light strongly at a wavelength of 340 nm, a property that makes it particularly useful for spectrophotometric assays.
This absorption is due to the electronic structure of NADH, which has specific chromophores that interact with UV light. NAD+, the oxidized form of NADH, does not absorb light at this wavelength, which allows for selective measurement of NADH concentration in a mixture.
In enzyme kinetics, monitoring the absorption of NADH at 340 nm can help track the progress of reactions where NADH is either consumed or produced. This is fundamental in understanding how enzymes like lactate dehydrogenase function and affect metabolic pathways.

spectrophotometry

Spectrophotometry is a technique used to measure how much light a chemical substance absorbs. A spectrophotometer is the instrument used for this measurement. It works by passing a beam of light through a sample and measuring the intensity of light that emerges compared to the intensity that entered it.
In the context of enzyme kinetics, such as with lactate dehydrogenase reactions, a spectrophotometer set to 340 nm is used to measure NADH concentration over time. The instrument provides a way to monitor changes in absorbance, which correspond to changes in the concentration of absorbing species (e.g., NADH).
This technique is invaluable in biochemical studies as it allows researchers to derive kinetic parameters such as reaction rates and enzyme efficiency. By obtaining absorbance data at different time points, we can follow the conversion of substrates to products in real time.

reaction rate calculation

Calculating the reaction rate involves determining how fast a reactant is consumed or a product is formed over time. In the case of the LDH-catalyzed reaction, we monitor the decrease in NADH absorbance at 340 nm.
Using the Beer-Lambert Law, which states that absorbance (A) is directly proportional to concentration (c):
\(A = \varepsilon \cdot l \cdot c\)
where \(\varepsilon\) is the molar absorptivity, \(l\) is the path length of the cuvette, and \(c\) is the concentration.
By measuring the change in absorbance (\(\Delta A\)) over time (\(\Delta t\)), the reaction rate can be calculated as:
\(rate = \dfrac{\Delta A}{\Delta t}\cdot \dfrac{1}{\varepsilon \cdot l}\)
This equation allows us to convert absorbance changes into concentration changes, giving a clear picture of how fast the reaction is proceeding. This method is not only accurate but also practical for a wide range of biochemical experiments.